Methods and compositions for the identification of antibiotics that are not susceptible to antibiotic resistance

ABSTRACT

Compositions and methods are provided to identify functional mutant ribosomes that may be used as drug targets. The compositions and methods allow isolation and analysis of mutations that would normally be lethal and allow direct selection of rRNA mutants with predetermined levels of ribosome function. The compositions and methods of the present invention may be used to identify antibiotics to treat a large number of human pathogens through the use of genetically engineered rRNA genes from a variety of species. The invention further provides novel plasmid constructs to be used in the methods of the invention.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/436,349, filed on May 18, 2006, now U.S. Pat. No. 7,709,196;which is a divisional of U.S. patent application Ser. No. 10/612,224,filed on Jul. 1, 2003, now U.S. Pat. No. 7,081,341; which claimspriority from U.S. provisional patent application Ser. No. 60/393,237,filed on Jul. 1, 2002, and U.S. provisional patent application Ser. No.60/452,012, filed on Mar. 5, 2003, all of which are expresslyincorporated by reference.

BACKGROUND OF THE INVENTION

Ribosomes are composed of one large and one small subunit containingthree or four RNA molecules and over fifty proteins. The part of theribosome that is directly involved in protein synthesis is the ribosomalRNA (rRNA). The ribosomal proteins are responsible for folding the rRNAsinto their correct three-dimensional structures. Ribosomes and theprotein synthesis process are very similar in all organisms. Onedifference between bacteria and other organisms, however, is the waythat ribosomes recognize mRNA molecules that are ready to be translated.In bacteria, this process involves a base-pairing interaction betweenseveral nucleotides near the beginning of the mRNA and an equal numberof nucleotides at the end of the ribosomal RNA molecule in the smallsubunit. The mRNA sequence is known as the Shine-Dalgarno (SD) sequenceand its counterpart on the rRNA is called the Anti-Shine-Dalgarno (ASD)sequence.

There is now extensive biochemical, genetic and phylogenetic evidenceindicating that rRNA is directly involved in virtually every aspect ofribosome function (Garrett, R. A., et al. (2000) The Ribosome:Structure, Function, Antibiotics, and Cellular Interactions. ASM Press,Washington, D.C.). Genetic and functional analyses of rRNA mutations inE. coli and most other organisms have been complicated by the presenceof multiple rRNA genes and by the occurrence of dominant lethal rRNAmutations. Because there are seven rRNA operons in E. coli, thephenotypic expression of rRNA mutations may be affected by the relativeamounts of mutant and wild-type ribosomes in the cell. Thus, detectionof mutant phenotypes can be hindered by the presence of wild-typeribosomes. A variety of approaches have been designed to circumventthese problems.

One common approach uses cloned copies of a wild-type rRNA operon(Brosius, J., et al. (1981) Plasmid 6: 112-118; Sigmund, C. D. et al.(1982) Proc. Natl. Acad. Sci. U.S.A. 79: 5602-5606). Several groups haveused this system to detect phenotypic differences caused by a high levelof expression of mutant ribosomes. Recently, a strain of E. coli wasconstructed in which the only supply of ribosomal RNA was plasmidencoded (Asai, T., (1999) J. Bacteriol. 181: 3803-3809). This system hasbeen used to study transcriptional regulation of rRNA synthesis, as wellas ribosomal RNA function (Voulgaris, J., et al. (1999) J. Bacteriol.181: 4170-4175; Koosha, H., et al. (2000) RNA. 6: 1166-1173; Sergiev, P.V., et al. (2000) J. Mol. Biol. 299: 379-389; O'Connor, M. et al. (2001)Nuci. Acids Res. 29: 1420-1425; O'Connor, M., et al. (2001) Nucl. AcidsRes. 29: 710-715; Vila-Sanjurjo, A. et al. (2001) J. Mol. Biol. 308:457-463); Morosyuk S. V., et al. (2000) J. Mol. Biol. 300 (1):113-126;Morosyuk S. V., et al. (2001) J. Mol. Biol. 307 (1):197-210; andMorosyuk S. V., et al. (2001) J. Mol. Biol. 307 (1):211-228. Hui et al.showed that mRNA could be directed to a specific subset ofplasmid-encoded ribosomes by altering the message binding site (MBS) ofthe ribosome while at the same time altering the ribosome binding site(RBS) of an mRNA (Hui, A., et al. (1987) Methods Enzymol. 153: 432-452).

Although each of the above methods has contributed significantly to theunderstanding of rRNA function, progress in this field has been hamperedboth by the complexity of translation and by difficulty in applyingstandard genetic selection techniques to these systems.

Resistance to antibiotics, a matter of growing concern, is caused partlyby antibiotic overuse. According to a study published by the Journal ofthe American Medical Association in 2001, between 1989 to 1999 Americanadults made some 6.7 million visits a year to the doctor for sorethroat. In 73% of those visits, the study found, the patient was treatedwith antibiotics, though only 5%-17% of sore throats are caused bybacterial infections, the only kind that respond to antibiotics.Macrolide antibiotics in particular are becoming extremely popular fortreatment of upper respiratory infections, in part because of theirtypically short, convenient course of treatment. Research has linkedsuch vast use to a rise in resistant bacteria and the recent developmentof multiple drug resistance has underscored the need for antibioticswhich are highly specific and refractory to the development of drugresistance.

Microorganisms can be resistant to antibiotics by four mechanisms.First, resistance can occur by reducing the amount of antibiotic thataccumulates in the cell. Cells can accomplish this by either reducingthe uptake of the antibiotic into the cell or by pumping the antibioticout of the cell. Uptake mediated resistance often occurs, because aparticular organism does not have the antibiotic transport protein onthe cell surface or occasionally when the constituents of the membraneare mutated in a way that interferes with transport of the antibioticinto a cell. Uptake mediated resistance is only possible in instanceswhere the drug gains entry through a nonessential transport molecule.Efflux mechanisms of antibiotic resistance occur via transporterproteins. These can be highly specific transporters that transport aparticular antibiotic, such as tetracycline, out of the cell or they canbe more general transporters that transport groups of molecules withsimilar characteristics out of the cell. The most notorious example of anonspecific transporter is the multidrug resistance transporter (MDR).

Inactivating the antibiotic is another mechanism by which microorganismscan become resistant to antibiotics. Antibiotic inactivation isaccomplished when an enzyme in the cell chemically alters the antibioticso that it no longer binds to its intended target. These enzymes areusually very specific and have evolved over millions of years, alongwith the antibiotics that they inactivate. Examples of antibiotics thatare enzymatically inactivated are penicillin, chloramphenicol, andkanamycin.

Resistance can also occur by modifying or overproducing the target site.The target molecule of the antibiotic is either mutated or chemicallymodified so that it no long binds the antibiotic. This is possible onlyif modification of the target does not interfere with normal cellularfunctions. Target site overproduction is less common but can alsoproduce cells that are resistant to antibiotics.

Lastly, target bypass is a mechanism by which microorganisms can becomeresistant to antibiotics. In bypass mechanisms, two metabolic pathwaysor targets exist in the cell and one is not sensitive to the antibiotic.Treatment with the antibiotic selects cells with more reliance on thesecond, antibiotic-resistant pathway.

Among these mechanisms, the greatest concern for new antibioticdevelopment is target site modification. Enzymatic inactivation andspecific transport mechanisms require the existence of a substratespecific enzyme to inactivate or transport the antibiotic out of thecell. Enzymes have evolved over millions of years in response tonaturally occurring antibiotics. Since microorganisms cannotspontaneously generate new enzymes, these mechanisms are unlikely topose a significant threat to the development of new syntheticantibiotics. Target bypass only occurs in cells where redundantmetabolic pathways exist. As understanding of the MDR transportersincreases, it is increasingly possible to develop drugs that are nottransported out of the cell by them. Thus, target site modificationposes the greatest risk for the development of antibiotic resistance fornew classes of antibiotic and this is particularly true for thoseantibiotics that target ribosomes. The only new class of antibiotics inthirty-five years, the oxazolidinones, is a recent example of anantibiotic that has been compromised because of target sitemodification. Resistant strains containing a single mutation in rRNAdeveloped within seven months of its use in the clinical settings.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods which may beused to identify antibiotics that are not susceptible to the developmentof antibiotic resistance. In particular, rRNA genes from E. coli andother disease causing organisms are genetically engineered to allowidentification of functional mutant ribosomes that may be used as drugtargets, e.g., to screen chemical and peptide libraries to identifycompounds that bind to all functional mutant ribosomes but do not bindto human ribosomes. Antibiotics that recognize all biologically activeforms of the target molecule and are therefore not susceptible to thedevelopment of drug resistance by target site modification are thusidentified.

The invention provides plasmid constructs comprising an rRNA gene havinga mutant ASD sequence set forth in FIGS. 12 (SEQ ID NOS:24-47), 13 (SEQID NOS:48-61), 15 (SEQ ID NOS:62-111), and 16 (SEQ ID NOS:112-159), atleast one mutation in the rRNA gene, and a genetically engineered genewhich encodes a selectable marker having a mutant SD sequence set forthin FIGS. 12, 13, 15, and 16. The mutant SD-ASD sequences are mutuallycompatible pairs and therefore permit translation of only the mRNAcontaining the compatible mutant SD sequence, i.e., translation of theselectable marker. In one embodiment, the selectable marker is chosenfrom the group consisting of chloramphenicol acetyltransferase (CAT),green fluorescent protein (GFP), or both CAT and GFP. In anotherembodiment, the DNA sequence encoding the rRNA gene is under the controlof an inducible promoter.

The rRNA gene may be selected from a variety of species, therebyproviding for the identification of functional mutant ribosomes that maybe used as drug targets to identify drug candidates that are effectiveagainst the selected species. Examples of species include, withoutlimitation, Mycobacterium tuberculosis (tuberculosis), Pseudomonasaeruginosa (multidrug resistant nosocomial infections), Salmonella typhi(typhoid fever), Yersenia pestis (plague), Staphylococcus aureus(multidrug resistant infections causing impetigo, folliculitis,abcesses, boils, infected lacerations, endocarditis, meningitis, septicarthritis, pneumonia, osteomyelitis, and toxic shock), Streptococcuspyogenes (streptococcal sore throat, scarlet fever, impetigo,erysipelas, puerperal fever, and necrotizing fascitis), Enterococcusfaecalis (vancomycin resistant nosocomial infections, endocarditis, andbacteremia), Chlamydia trachomatis (lymphogranuloma venereum, trachomaand inclusion conjunctivitis, nongonococcal urethritis, epididymitis,cervicitis, urethritis, infant pneumonia, pelvic inflammatory diseases,Reiter's syndrome (oligoarthritis) and neonatal conjunctivitis),Saccharomyces cerevesiae, Candida albicans, and trypanosomes. In oneembodiment, the rRNA gene is from Mycobacterium tuberculosis (see, e.g.,Example 6 and FIG. 17).

In still other embodiments of the invention, the rRNA genes aremitochondrial rRNA genes, i.e., eukaryotic rRNA genes (e.g., humanmitochondrial rRNA genes).

The plasmid constructs of the invention, such as the plasmid constructsset forth in FIGS. 22-26, may include novel mutant ASD and SD sequencesset forth herein. In particular, the present invention provides novelmutant ASD sequences and novel mutant SD sequences, set forth in FIGS.12, 13, 15, and 16, which may be used in the plasmid constructs andmethods of the invention. The mutant ASD and mutant SD sequences may beused as mutually compatible pairs (see FIGS. 12, 13, 15, and 16). Itwill be appreciated that the mutually compatible pairs of mutant ASD andSD sequences interact as pairs in the form of RNA and permit translationof only the mRNAs containing the compatible mutant SD sequence.

In another aspect, the present invention provides a plasmid comprisingan E. coli 16S rRNA gene having a mutant ASD sequence, at least onemutation in said 16S rRNA gene, and a genetically engineered gene whichencodes a selectable marker, e.g., GFP, having a mutant SD sequence. Inanother embodiment, the 16S rRNA gene is from a species other than E.coli. In one embodiment, the mutant ASD sequence is selected from thesequences set forth in FIGS. 12, 13, 15, and 16. In another embodiment,the mutant SD sequence is selected from the sequences set forth in FIGS.12, 13, 15, and 16. In yet another embodiment, the mutant ASD sequenceand the mutant SD sequence are in mutually compatible pairs (see FIGS.12, 13, 15, and 16). Each mutually compatible mutant SD and mutant ASDpair permits translation by the selectable marker.

In one embodiment, the invention features a cell comprising a plasmid ofthe invention. In another embodiment, the cell is a bacterial cell.

In one embodiment, the invention provides a method for identifyingfunctional mutant ribosomes comprising:

(a) transforming a host cell with a plasmid comprising an rRNA genehaving a mutant ASD sequence, at least one mutation in said rRNA gene,and a genetically engineered gene which encodes a selectable markerhaving a mutant SD sequence, wherein the mutant ASD and mutant SDsequences are a mutually compatible pair;

(b) isolating cells via the selectable marker; and

(c) identifying the rRNA from the cells from step (b), therebyidentifying functional mutant ribosomes.

In another embodiment, the invention features a method for identifyingfunctional mutant ribosomes comprising:

(a) transforming a host cell with a plasmid comprising an E. coli 16SrRNA gene having a mutant ASD sequence, at least one mutation in said16S rRNA gene, and a genetically engineered gene which encodes GFPhaving a mutant SD sequence wherein the mutant ASD and mutant SDsequences are a mutually compatible pair;

(b) isolating cells via the GFP; and

(c) identifying the rRNA from the cells from step (b), therebyidentifying functional mutant ribosomes.

In yet another embodiment, the invention features a method foridentifying functional mutant ribosomes that may be suitable as drugtargets comprising:

(a) transforming a host cell with a plasmid comprising an rRNA genehaving a mutant ASD sequence, at least one mutation in said rRNA gene,and a genetically engineered gene which encodes a selectable markerhaving a mutant SD sequence, wherein the mutant ASD and mutant SDsequences are a mutually compatible pair;

(b) isolating cells via the selectable marker;

(c) identifying and sequencing the rRNA from the cells from step (b),thereby identifying regions of interest;

(d) selecting regions of interest from step (c);

(e) mutating the regions of interest from step (d);

(f) inserting the mutated regions of interest from step (e) into aplasmid comprising an rRNA gene having a mutant ASD sequence and agenetically engineered gene which encodes a selectable marker having amutant SD sequence, wherein the mutant ASD and mutant SD sequences are amutually compatible pair;

(g) transforming a host cell with the plasmid from step (f);

(h) isolating cells of step (g) via the selectable marker; and

(i) identifying the rRNA from step (h), thereby identifying functionalmutant ribosomes that may be suitable as drug targets.

In a further embodiment, the invention provides a method for identifyingfunctional mutant ribosomes that may be suitable as drug targetscomprising:

(a) transforming a host cell with a plasmid comprising an E. coli 16SrRNA gene having a mutant ASD sequence, at least one mutation in said16S rRNA gene, and a genetically engineered gene which encodes GFPhaving a mutant SD sequence wherein the mutant ASD and mutant SDsequences are a mutually compatible pair;

(b) isolating cells via the GFP;

(c) identifying and sequencing the rRNA from the cells from step (b),thereby identifying regions of interest;

(d) selecting the regions of interest from step (c);

(e) mutating the regions of interest from step (d);

(f) inserting the mutated regions of interest from step (e) into aplasmid comprising an E. coli 16S rRNA gene having a mutant ASD sequenceand a genetically engineered gene which encodes GFP having a mutant SDsequence, wherein the mutant ASD and mutant SD sequences are a mutuallycompatible pair;

(g) transforming a host cell with the plasmid from step (f);

(h) isolating cells of step (g) via the GFP; and

(i) identifying the rRNA from step (h), thereby identifying functionalmutant ribosomes that may be suitable as drug targets.

In one embodiment, the invention features a method for identifying drugcandidates comprising:

(a) transforming a host cell with a plasmid comprising an rRNA genehaving a mutant ASD sequence, at least one point mutation in said rRNAgene, and a genetically engineered gene which encodes a selectablemarker having a mutant SD sequence, wherein the mutant ASD and mutant SDsequences are a mutually compatible pair;

(b) isolating cells via the selectable marker;

(c) identifying and sequencing the rRNA from step (b) to identify theregions of interest;

(d) selecting the regions of interest from step (c);

(e) mutating the regions of interest from step (d);

(f) inserting the mutated regions of interest from step (e) into aplasmid comprising an rRNA gene having a mutant ASD sequence and agenetically engineered gene which encodes a selectable marker having amutant SD sequence, wherein the mutant ASD and mutant SD sequences are amutually compatible pair;

(g) transforming a host cell with the plasmid from step (f);

(h) isolating the cells from step (g) via the selectable marker;

(i) identifying the rRNA from step (h) to identify the functional mutantribosomes;

(j) screening drug candidates against functional mutant ribosomes fromstep (i);

(k) identifying the drug candidates from step (j) that bound to thefunctional mutant ribosomes from step (i);

(l) screening the drug candidates from step (k) against human rRNA; and

(m) identifying the drug candidates from step (l) that do not bind tohuman rRNA, thereby identifying drug candidates.

In one embodiment, the invention provides a method for identifying drugcandidates comprising:

(a) transforming a host cell with a plasmid comprising an E. coli 16SrRNA gene having a mutant ASD sequence, at least one point mutation insaid 16S rRNA gene, and a genetically engineered gene which encodes GFPhaving a mutant SD sequence, wherein the mutant ASD and mutant SDsequences are a mutually compatible pair;

(b) isolating the cells via the selectable marker;

(c) identifying and sequencing the rRNA from step (b) to identify theregions of interest;

(d) selecting the regions of interest from step (c);

(e) mutating the regions of interest from step (d);

(f) inserting the mutated regions of interest from step (e) into aplasmid comprising an E. coli 16S rRNA gene having a mutant ASD sequenceand a genetically engineered gene which encodes GFP having a mutant SDsequence, wherein the mutant ASD and mutant SD sequences are a mutuallycompatible pair;

(g) transforming a host cell with the plasmid from step (f);

(h) isolating cells from step (g) via the selectable marker;

(i) identifying the rRNA from step (h) to identify the functional mutantribosomes;

(j) screening drug candidates against the functional mutant ribosomesfrom step (i);

(k) identifying the drug candidates from step (j) that bound to thefunctional mutant ribosomes from step (i);

(l) screening the drug candidates from step (k) against human 16S rRNA;and

(m) identifying the drug candidates from step (l) that do not bind tothe human 16S rRNA, thereby identifying drug candidates.

It will be appreciated that the rRNA gene used in the methods of thepresent invention may be from the 16S rRNA, 23S rRNA, and 55S rRNA gene.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the plasmid construct pRNA123. The locations of specificsites in pRNA123 are as follows: the 16S rRNA E. coli rrnB operoncorresponds to nucleic acids 1-1542; the 16S MBS (message bindingsequence) GGGAU corresponds to nucleic acids 1536-1540; the 16S-23Sspacer region corresponds to nucleic acids 1543-1982; the 23S rRNA of E.coli rrnB operon corresponds to nucleic acids 1983-4886; the 23S-5Sspacer region corresponds to nucleic acids 4887-4982; the 5S rRNA of E.coli rrnB operon corresponds to nucleic acids 4983-5098; the terminatorT1 of E. coli rrnB operon corresponds to nucleic acids 5102-5145; theterminator T2 of E. coli rrnB operon corresponds to nucleic acids5276-5305; the bla (β-lactamase; ampicillin resistance) corresponds tonucleic acids 6575-7432; the replication origin corresponds to nucleicacids 7575-8209; the rop (Rop protein) corresponds to nucleic acids8813-8622; the GFP corresponds to nucleic acids 10201-9467; the GFP RBS(ribosome binding sequence) AUCCC corresponds to nucleic acids10213-10209; the trp^(c) promoter corresponds to nucleic acids10270-10230; the trp^(c) promoter corresponds to nucleic acids10745-10785; the CAT RBS AUCCC corresponds to nucleic acids 10802-10806;the cam (chloramphenicol acetyltransferase: CAT) corresponds to nucleicacids 10814-11473; the lacl^(q) promoter corresponds to nucleic acids11782-11859; the lacl^(q) (lac repressor) corresponds to nucleic acids11860-12942; and the lacUV5 promoter corresponds to nucleic acids12985-13026.

FIG. 2 depicts a scheme for construction of pRNA9. The abbreviations inFIG. 2 are defined as follows: Ap^(r), ampicillin resistance; cam, CATgene; lacl^(q), lactose repressor; PlacUV5, lacUV5 promoter; Ptrp^(c),constitutive trp promoter. The restriction sites used are alsoindicated.

FIG. 3 depicts an autoradiogram of sequencing gels with pRNA8-rMBS-rRBS.The mutagenic MBS and RBS are shown: B 5 C, G, T; D 5 A, G, T; H 5 A, C,T; V 5 A, C, G. The start codon of cam and the 39 end of 16S rRNA areindicated. Panel A depicts the RBS of the CAT gene. Panel B depicts theMBS of the 16S rRNA gene.

FIG. 4 depicts a graph of the effect of MBSs on growth. Theabbreviations in FIG. 4 are defined as follows: pBR322; vector: pRNA6;RBS 5 GUGUG, MBS 5 CACAC: pRNA9; RBS 5 GGAGG (wt), MBS 5 CCUCC (wt): andClone IX24; RBS 5 AUCCC, MBS 5 GGGAU.

FIG. 5 depicts a scheme for construction of pRNA122. The abbreviationsin FIG. 5 are defined as follows: Ap^(r), ampicillin resistance; cam,CAT gene; lacl^(q), lactose repressor; PlacUV5, lacUV5 promoter;Ptrp^(c), constitutive trp promoter; N 5 A, C, G, and T. The fournucleotides mutated are underlined and the restriction sites used areindicated.

FIG. 6 depicts a plasmid-derived ribosome distribution and CAT activity.Cultures were induced (or not) in early log phase (as shown in FIG. 4)and samples were withdrawn for CAT assay and total RNA preparation atthe points indicated. Open squares represent the percent plasmid-derivedrRNA in uninduced cells. Closed squares represent the percentplasmid-derived rRNA in induced cells. Open circles represent CATactivity in uninduced cells. Closed circles represent CAT activity ininduced cells.

FIG. 7 depicts a scheme for construction of single mutations atpositions 516 or 535. The abbreviations in FIG. 7 are defined asfollows: Ap^(r), ampicillin resistance; cam, CAT gene; lacl^(q), lactoserepressor; PlacUV5, lacUV5 promoter; Ptrp^(c), constitutive trppromoter. C516 was substituted to V (A, C, or G) and A535 wassubstituted to B (C, G, or T,) in pRNA122 and the restriction sites thatwere used are also indicated.

FIG. 8 depicts the functional analysis of mutations constructed atpositions 516 and 535 of 16S rRNA in pRNA122. Nucleotide identities areindicated in the order of 516:535 and mutations are underlined. pRNA122containing the wild-type MBS (wt. MBS) was used as a negative control toassess the degree of MIC and the level of CAT activity due to CAT mRNAtranslation by wild-type ribosomes. Standard error of the mean is usedto indicate the range of the assay results.

FIG. 9 depicts a description and use of oligodeoxynucleotides (SEQ IDNOS:6-23). Primer binding sites are indicated by the number ofnucleotides from the 5′ nucleotide of the coding region. Negativenumbers indicate binding sites 5′ to the coding region.

FIG. 10 describes several plasmids used in Example 4.

FIG. 11 depicts the specificity of the selected recombinants. Theconcentrations of chloramphenicol used are indicated and the unit of MICis micrograms of chloramphenicol/mL.

FIG. 12 depicts novel mutant ASD sequences and novel mutant SD sequencesof the present invention (SEQ ID NOS:24-47). FIG. 12 also shows asequence analysis of chloramphenicol resistant isolates. The mutatednucleotides are underlined and potential duplex formations are boxed.CAT activity was measured twice for each culture and the unit is CPM/0.1μL of culture/OD600. Induction was measured by dividing CAT activity ininduced cells with CAT activity in uninduced cells. A-1 indicates noinduction, while a +1 indicates induction with 1 mM IPTG.

FIG. 13 depicts novel mutant ASD sequences and novel mutant SD sequencesof the present invention (SEQ ID NOS:48-61). FIG. 13 also shows asequence analysis of CAT mRNA mutants. Potential duplex formations areboxed and the mutated nucleotides are underlined. The start codon (AUG)is in bold. A-1 indicates no induction, while a +1 indicates inductionwith 1 mM IPTG.

FIG. 14 depicts the effect of Pseudouridine516 Substitutions on subunitassembly. The percent plasmid-derived 30S data are presented as thepercentage of the total 30S in each peak and in crude ribosomes.

FIG. 15 depicts novel mutant ASD sequences and novel mutant SD sequencesof the present invention (SEQ ID NOS:62-111).

FIG. 16 depicts novel mutant ASD sequences and novel mutant SD sequencesof the present invention (SEQ ID NOS:112-159).

FIG. 17 depicts a hybrid construct. This hybrid construct contains a 16SrRNA from Mycobacterium tuberculosis. The specific sites on the hybridconstruct are as follows: the part of rRNA from E. coli rrnB operoncorresponds to nucleic acids 1-931; the part of 16S rRNA fromMycobacterium tuberculosis rrn operon corresponds to nucleic acids932-1542; the 16S MBS (message binding sequence) GGGAU corresponds tonucleic acids 1536-1540; the terminator T1 of E. coli rrnB operoncorresponds to nucleic acids 1791-1834; the terminator T2 of E. colirrnB operon corresponds to nucleic acids 1965-1994; the replicationorigin corresponds to nucleic acids 3054-2438; the bla (β-lactamase;ampicillin resistance) corresponds to nucleic acids 3214-4074; the GFPcorresponds to nucleic acids 5726-4992; the GFP RBS (ribosome bindingsequence) AUCCC corresponds to nucleic acids 5738-5734; the trp^(c)promoter corresponds to nucleic acids 5795-5755; the trp^(c) promotercorresponds to nucleic acids 6270-6310; the CAT RBS (ribosome bindingsequence) AUCCC corresponds to nucleic acids 6327-6331; the cam(chloramphenicol acetyltransferase; CAT) corresponds to nucleic acids6339-6998; the lacl^(q) promoter corresponds to nucleic acids 7307-7384;the lacl^(q) (lac repressor) corresponds to nucleic acids 7385-8467; andthe lacUV5 promoter corresponds to nucleic acids 8510-8551.

FIG. 18 depicts a plasmid map of pRNA122.

FIG. 19 depicts a table of sequences and MICs of functional mutants (SEQID NOS:160-238). Sequences are ranked by the minimum inhibitoryconcentration (“MIC”) of chloramphenicol required to fully inhibitgrowth of cells expressing the mutant ribosomes. The nucleotidesequences (“Nucleotide sequence”) are the 790 loop sequences selectedfrom the pool of functional, randomized mutants. Mutations areunderlined. The number of mutations (“Number of mutations”) in eachmutant sequence are indicated, as well as the number of occurrences(“Number of occurrences”) which represents the number of clones with theindicated sequence. The sequence and activity of the unmutated control,pRNA122 (WT, wild-type) is depicted in the first row of FIG. 19, inwhich the MIC is 600 μg/ml.

FIG. 20 depicts the 790-loop sequence variation. In the consensussequence R=A or G; N=A, C, G or U; M=A or C; H=A, C or U; W=A or U; Y=Cor U; Δ=deletion; and underlined numbers indicate the wild-type E. colisequence.

FIG. 21 depicts functional and thermodynamic analysis of positions 787and 795. Mutations have been underlined and “n.d.” represents notdetermined. FIG. 21 shows site-directed mutations (“Nucleotide”) thatwere constructed using PCR, as described for the random mutants, exceptthat the mutagenic primers contained substitutions corresponding only topositions 787 and 795. In order to determine ribosome function (“MeanCAT activity”), each strain was grown and assayed for CAT activity atleast twice, the data were averaged, and presented as percentages of theunmutated control, pRNA122+ the standard error of the mean. The ratio ofplasmid to chromosome-derived rRNA in 30S and 70 S ribosomes (“% Mutant30S in 30S peak/70S peak”) was determined by primer extension. Cultureswere grown and assayed at least twice and the mean values are presentedas a percentage of the total 30S in each peak ±the standard error of themean. Thermodynamic parameters (“Thermodynamics”) are for thehigher-temperature transition of model oligonucleotides and are theaverage of results for four or five different oligomer concentrations.Standard errors for the ΔG° 37 are ±5% (1 kcal=4184 J). Errors in T_(m)are estimated as ±1° C. All solutions were at pH 7.

FIG. 22 depicts the DNA sequence of pRNA8 (SEQ ID NO:1).

FIG. 23 depicts the DNA sequence of pRNA122 (SEQ ID NO:2).

FIG. 24 depicts the DNA sequence of pRNA123 (SEQ ID NO:3).

FIG. 25 depicts the DNA sequence of pRNA123 Mycobacterium tuberculosis-2(pRNA123 containing a hybrid of E. coli and Mycobacterium tuberculosis16S rRNA genes) (SEQ ID NO:4).

FIG. 26 depicts the DNA sequence of pRep-Mycobacterium tuberculosis-2(containing a puc19 derivative containing the rRNA operon from pRNA122;however, the 23S and 5S rRNA genes are deleted) (SEQ ID NO:5).

FIGS. 2-14 may be found in Lee, K., et al. Genetic Approaches toStudying Protein Synthesis Effects of Mutations at Pseudouridine 516 andA535 in Escherichia coli 16S rRNA. Symposium: Translational Control: AMechanistic Perspective at the Experimental Biology 2001 Meeting (2001);and FIGS. 18-21 may be found in Lee, K. et al., J. Mol. Biol. 269:732-743 (1997), all of which are expressly incorporated by referenceherein.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods are provided to identify functional mutantribosomes suitable as drug targets. The compositions and methods allowisolation and analysis of mutations that would normally be lethal andallow direct selection of rRNA mutants with predetermined levels ofribosome function. The compositions and methods of the present inventionmay be used to identify antibiotics to treat generally and/orselectively human pathogens.

According to one embodiment of the invention, a functional genomicsdatabase for rRNA genes of a variety of species may be generated. Inparticular, the rRNA gene is randomly mutated using a generalizedmutational strategy. A host cell is then transformed with a mutagenizedplasmid of the invention comprising: an rRNA gene having a mutant ASDsequence, the mutated rRNA gene, and a genetically engineered gene whichencodes a selectable marker having a mutant SD sequence. The selectablemarker gene, such as CAT, may be used to select mutants that arefunctional, e.g., by plating the transformed cells onto growth mediumcontaining chloramphenicol. The mutant rRNA genes contained in eachplasmid DNA of the individual clones from each colony are selected andcharacterized. The function of each of the mutant rRNA genes is assessedby measuring the amount of an additional selectable marker gene, such asGFP, produced by each clone upon induction of the rRNA operon. Afunctional genomics database may thus be assembled, which contains thesequence and functional data of the functional mutant rRNA genes. Inparticular, functionally important regions of the rRNA gene that willserve as drug targets are identified by comparing the sequences of thefunctional genomics database and correlating the sequence with theamount of GFP protein produced.

In another embodiment, the nucleotides in the functionally importanttarget regions identified in the above methods may be simultaneouslyrandomly mutated, e.g., by using standard methods of molecularmutagenesis, and cloned into a plasmid of the invention to form aplasmid pool containing random mutations at each of the nucleotidepositions in the target region. The resulting pool of plasmidscontaining random mutations is then used to transform cells, e.g., E.coli cells, and form a library of clones, each of which contains aunique combination of mutations in the target region. The library ofmutant clones are grown in the presence of IPTG to induce production ofthe mutant rRNA genes and a selectable marker is used, such as CAT, toselect clones of rRNA mutants containing nucleotide combinations of thetarget region that produce functional ribosomes. The rRNA genesproducing functional ribosomes are sequenced and may be incorporatedinto a database.

In yet another embodiment, a series of oligonucleotides may besynthesized that contain the functionally-important nucleotides andnucleotide motifs within the target region and may be used tosequentially screen compounds and compound libraries to identifycompounds that recognize (bind to) the functionally important sequencesand motifs. The compounds that bind to all of the oligonucleotides arethen counterscreened against oligonucleotides and/or other RNAcontaining molecules to identify drug candidates. Drug candidatesselected by the methods of the present invention are thus capable ofrecognizing all of the functional variants of the target sequence, i.e.,the target cannot be mutated in a way that the drug cannot bind, withoutcausing loss of function to the ribosome.

In still another embodiment, after the first stage mutagenesis of theentire rRNA is performed using techniques known in the art, e.g.,error-prone PCR mutagenesis, the mutants are analyzed to identifyregions within the rRNA that are important for function. These regionsare then sorted based on their phylogenetic conservation, as describedherein, and are then used for further mutagenesis.

Ribosomal RNA sequences from each species are different and the moreclosely related two species are, the more their rRNAs are alike. Forinstance, humans and monkeys have very similar rRNA sequences, buthumans and bacteria have very different rRNA sequences. Thesedifferences may be utilized for the development of very specific drugswith a narrow spectrum of action and also for the development ofbroad-spectrum drugs that inhibit large groups of organisms that areonly distantly related, such as all bacteria.

In another embodiment, the functionally important regions identifiedabove are divided into groups based upon whether or not they occur inclosely related groups of organisms. For instance, some regions of rRNAare found in all bacteria but not in other organisms. Other areas ofrRNA are found only in closely related groups of bacteria, such as allof the members of a particular species, e.g., members of the genusMycobacterium or Streptococcus.

In a further embodiment, the regions found in very large groups oforganisms, e.g., all bacteria or all fungi, are used to developbroad-spectrum antibiotics that may be used to treat infections from alarge number of organisms within that group. The methods of the presentinvention may be performed on these regions and functional mutantribosomes identified. These functional mutant ribosomes may be screened,for example, with compound libraries.

In yet another embodiment, regions that are located only in relativelysmall groups of organisms, such as all members of the genusStreptococcus or all members of the genus Mycobacterium, may be used todesign narrow spectrum antibiotics that will only inhibit the growth oforganisms that fall within these smaller groups. The methods of thepresent invention may be performed on these regions and functionalmutant ribosomes identified. These functional mutant ribosomes will bescreened, e.g., compound libraries.

The invention provides novel plasmid constructs, e.g. pRNA123 (FIGS. 1and 24). The novel plasmid constructs of the present invention employnovel mutant ASD and mutant SD sequences set forth in FIGS. 12, 13, 15and 16. The mutant ASD and mutant SD sequences may be used as mutuallycompatible pairs (see FIGS. 12, 13, 15 and 16). It will be appreciatedthat the mutually compatible pairs of mutant ASD and SD sequencesinteract as pairs in the form of RNA, to permit translation of only themRNAs containing the altered SD sequence.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living cell substantiallyonly when an inducer which corresponds to the promoter is present in thecell.

As used herein, the term “mutation” includes an alteration in thenucleotide sequence of a given gene or regulatory sequence from thenaturally occurring or normal nucleotide sequence. A mutation may be asingle nucleotide alteration (e.g., deletion, insertion, substitution,including a point mutation), or a deletion, insertion, or substitutionof a number of nucleotides.

By the term “selectable marker” is meant a gene whose expression allowsone to identify functional mutant ribosomes.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA)and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, or culture medium,when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence set forth in FIGS. 12, 13, 15,and 16, or a portion thereof, can be isolated using standard molecularbiology techniques and the sequence information provided herein. Usingall or portion of the nucleic acid sequence set forth in FIGS. 12, 13,15, and 16 as a hybridization probe, the nucleic acid molecules of thepresent invention can be isolated using standard hybridization andcloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F.,and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of thesequence set forth in FIGS. 12, 13, 15, and 16 can be isolated by thepolymerase chain reaction (PCR) using synthetic oligonucleotide primersdesigned based upon the sequence set forth in FIGS. 12, 13, 15, and 16.

A nucleic acid of the invention can be amplified using cDNA, mRNA or,alternatively, genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to the nucleotide sequences of the present invention canbe prepared by standard synthetic techniques, e.g., using an automatedDNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence set forth in FIGS. 12, 13, 15, and 16, or aportion of any of these nucleotide sequences. A nucleic acid moleculewhich is complementary to the nucleotide sequence shown in FIGS. 12, 13,15, and 16, is one which is sufficiently complementary to the nucleotidesequence shown in FIGS. 12, 13, 15, and 16, such that it can hybridizeto the nucleotide sequence shown in FIGS. 12, 13, 15, and 16,respectively, thereby forming a stable duplex.

II. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid molecule of the presentinvention (or a portion thereof). As used herein, the term “vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked. One type of vector is a“plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel (1990) Methods Enzymol. 185:3-7.Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in many types of host cells and those whichdirect expression of the nucleotide sequence only in certain host cells(e.g., tissue-specific regulatory sequences). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d (Studieret al. (1990) Methods Enzymol. 185:60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn 10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S. (1990)Methods Enzymol. 185:119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector may be a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari et al. (1987) Embo J. 6:229-234), pMFa (Kurjanand Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987)Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (Invitrogen Corp, San Diego, Calif.).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example by the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379).

Another aspect of the invention pertains to host cells into which a thenucleic acid molecule of the invention is introduced. The terms “hostcell” and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. Other suitablehost cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

III. Uses and Methods of the Invention

The nucleic acid molecules described herein may be used in a plasmidconstruct, e.g. pRNA123, to carry out one or more of the followingmethods: (1) creation of a functional genomics database of the rRNAgenes generated by the methods of the present invention; (2) mining ofthe database to identify functionally important regions of the rRNA; (3)identification of functionally important sequences and structural motifswithin each target region; (4) screening compounds and compoundlibraries against a series of functional variants of the target sequenceto identify compounds that bind to all functional variants of the targetsequence; and (5) counterscreening the compounds against nontarget RNAs,such as human ribosomes or ribosomal RNA sequences.

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and Appendices, are incorporatedherein by reference.

SPECIFIC EXAMPLES Example 1 Identification of Mutant SD and Mutant ASDCombinations

It has been shown that by coordinately changing the SD and ASD, aparticular mRNA containing an altered SD could be targeted to ribosomescontaining the altered ASD. This and all other efforts to modify theASD, however, have proved lethal, as cells containing these mutationsdied within two hours after the genes containing them were activated.

Using random mutagenesis and genetic selection, mutant SD-ASDcombinations were screened in order to identify nonlethal SD-ASDcombinations. The mutant SD-ASD mutually compatible pairs are set forthin FIGS. 12, 13 15 and 16. The mutually compatible pairs of mutantsequences interact as pairs in the form of RNA. The novel mutant SD-ASDsequence combinations of the present invention permit translation ofonly the mRNAs containing the altered SD sequence.

Example 2 Construction of the pRNA123 Plasmid

A plasmid construct of the present invention identified as the pRNA123plasmid, is set forth in FIGS. 1 and 24. E. coli cells contain a singlechromosome with seven copies of the rRNA genes and all of the genes forthe ribosomal proteins. The plasmid, pRNA123, in the cell contains agenetically engineered copy of one of the rRNA genes from E. coli andtwo genetically engineered genes that are not normally found in E. coli,referred to herein as a “selectable markers.” One gene encodes theprotein chloramphenicol acetyltransferase (CAT). This protein renderscells resistant to chloramphenicol by chemically modifying theantibiotic. Another gene, the Green Fluorescent Protein (GFP), is alsoincluded in the system. GFP facilitates high-throughput functionalanalysis. The amount of green light produced upon irradiation withultraviolet light is proportional to the amount of GFP present in thecell.

Ribosomes from pRNA123 have an altered ASD sequence. Therefore, theribosomes can only translate mRNAs that have an altered SD sequence.Only two genes in the cell produce mRNAs with altered SD sequences thatmay be translated by the plasmid-encoded ribosomes: the CAT and GFPgene. Mutations in rRNA affect the ability of the resulting mutantribosome to make protein. The present invention thus provides a systemwhereby the mutations in the plasmid-encoded rRNA gene only affect theamount of GFP and CAT produced. A decrease in plasmid ribosome functionmakes the cell more sensitive to chloramphenicol and reduces the amountof green fluorescence of the cells. Translation of the other mRNAs inthe cell is unaffected since these mRNAs are translated only byribosomes that come from the chromosome. Hence, cells containingfunctional mutants may be identified and isolated via the selectablemarker.

Example 3 Genetic System for Functional Analysis of Ribosomal RNA

Identification of Functionally Important Regions of rRNA. Functionallyimportant regions of rRNA molecules that may be used as drug targetsusing a functional genomics approach may be identified through a seriesof steps. Namely, in step I.a., the entire rRNA gene is randomly mutatedusing error-prone PCR or another generalized mutational strategy. Instep I.b., a host cell is then transformed with a mutagenized plasmidcomprising: an rRNA gene having a mutant ASD sequence, at least onemutation in said rRNA gene, and a genetically engineered gene whichencodes a selectable marker having a mutant SD sequence, and productionof the rRNA genes from the plasmid are induced by growing the cells inthe presence of IPTG. In step I.c., the CAT gene is used to selectmutants that are functional by plating the transformed cells onto growthmedium containing chloramphenicol. In step I.d., individual clones fromeach of the colonies obtained in step I.c. are isolated. In step I.e.,the plasmid DNA from each of the individual clones from step I.d. isisolated. In step I.f., the rRNA genes contained in each of the plasmidsthat had been isolated in step I.e. are sequenced. In step I.g., thefunction of each of the mutants from step I.f. is assessed by measuringthe amount of GFP produced by each clone from step I.e. upon inductionof the rRNA operon. In step I.h., a functional genomics database isassembled containing the sequence and functional data from steps I.f.and I.g. In step I.i., functionally important regions of the rRNA genethat will serve as drug targets are identified. Functionally importantregions may be identified by comparing the sequences of all of thefunctional genomics database constructed in step I.g. and correlatingthe sequence with the amount of GFP protein produced. Contiguoussequences of three or more rRNA nucleotides, in which substitution ofthe nucleotides in the region produces significant loss of function,will constitute a functionally important region and therefore apotential drug target.

Isolation of Functional Variants of the Target Regions. A second aspectof the invention features identification of mutations of the target sitethat might lead to antibiotic resistance using a process termed,“instant evolution”, as described below. In step II.a., for a giventarget region identified in step I.i., each of the nucleotides in thetarget region is simultaneously randomly mutated using standard methodsof molecular mutagenesis, such as cassette mutagenesis or PCRmutagenesis, and cloned into the plasmid of step I.b. to form a plasmidpool containing random mutations at each of the nucleotide positions inthe target region. In step II.b., the resulting pool of plasmidscontaining random mutations from step II.a. is used to transform E. colicells and form a library of clones, each of which contains a uniquecombination of mutations in the target region. In step II.c., thelibrary of mutant clones from step II.b. is grown in the presence ofIPTG to induce production of the mutant rRNA genes. In step II.d., theinduced mutants are plated on medium containing chloramphenicol, and CATis used to select clones of rRNA mutants containing nucleotidecombinations of the target region that produce functional ribosomes. Instep II.e., the functional clones isolated in step II.d. are sequencedand GFP is used to measure ribosome function in each one. In step II.f.,the data from step II.e. are incorporated into a mutational database.

Isolation of Drug Leads. In step III.a., the database in step II.f. isanalyzed to identify functionally-important nucleotides and nucleotidemotifs within the target region. In step III.b., the information fromstep III.a. is used to synthesize a series of oligonucleotides thatcontain the functionally important nucleotides and nucleotide motifsidentified in step III.a. In step III.c., the oligonucleotides from stepIII.b. are used to sequentially screen compounds and compound librariesto identify compounds that recognize (bind to) the functionallyimportant sequences and motifs. In step III.d., compounds that bind toall of the oligonucleotides are counterscreened against oligonucleotidesand/or other RNA containing molecules to identify drug candidates. “Drugcandidates” are compounds that 1) bind to all of the oligonucleotidescontaining the functionally important nucleotides and nucleotide motifs,but do not bind to molecules that do not contain the functionallyimportant nucleotides and nucleotide motifs and 2) do not recognizehuman ribosomes. Drug candidates selected by the methods of the presentinvention therefore recognize all of the functional variants of thetarget sequence, i.e., the target cannot be mutated in a way that thedrug cannot bind, without causing loss of function to the ribosome.

Example 4 Genetic System for Studying Protein Synthesis

Materials and Methods

Reagents. All reagents and chemicals were as in Lee, K., et al. (1996)RNA 2: 1270-1285. PCR-directed mutagenesis was performed essentially bythe method of Higuchi, R. (1989) PCR Technology (Erlich, H. A., ed.),pp. 61-70. Stockton Press, New York, N.Y. The primers used in thepresent invention are listed in FIG. 9. The plasmids used in the presentinvention are listed in FIG. 10.

Bacterial strains and media. All plasmids were maintained and expressedin E. coli DH5 (supE44, hsdR17, recA1, endA1, gyrA96, thi-1 and relA1)(36). To induce synthesis of plasmid-derived rRNA from the lacUV5promoter, IPTG was added to a final concentration of 1 mM.Chloramphenicol acetyltransferase activity was determined essentially asdescribed by Nielsen et al. (1989) Anal. Biochem. 179: 19-23. Culturesfor CAT assays were grown in LB-Ap100. MIC were determined by standardmethods in microtiter plates as described in Lee, K., et al. (1997) J.Mol. Biol. 269: 732-743.

Primer extension. To determine the ratio of plasmid tochromosome-derived rRNA, pRNA104 containing cells growing in LB-Ap100were harvested at the time intervals indicated and total RNA wasextracted using the Qiagen RNeasy kit (Chatsworth, Calif.). The 30S,70S, and crude ribosomes were isolated from 200 mL of induced, plasmidcontaining cells by the method of Powers and Noller (Powers, T. et al.(1991) EMBO J. 10: 2203-2214). The purified RNA was analyzed by primerextension according to Sigmund, C. D., et al. (1988) Methods Enzymol.164: 673-690.

Experimental Procedures

Generation of pRNA9 construct. The initial construct, pRNA9, wasgenerated using the following methods. Plasmid pRNA9 contains a copy ofthe rrnB operon from pKK3535 under transcriptional regulation of thelacUV5 promoter; this well-characterized promoter is not subject tocatabolic repression and is easily and reproducibly inducible withisopropyl-β-D-thiogalactoside (IPTG). To minimize transcription in theabsence of inducer, PCR was used to amplify and subclone the lacrepressor variant, lacl^(q) (Calos, M. P. (1978) Nature 274: 762-765)from pSPORT1 (Life Technologies, Rockville, Md.). The chloramphenicolacetyltransferase gene (cam) is present and transcribed constitutivelyfrom a mutant tryptophan promoter, trp^(c) (De Boer, H. A., et al.(1983) Proc. Natl. Acad. Sci. U.S.A. 80: 21-25; Hui, A., et al. (1987)Proc. Natl. Acad. Sci. U.S.A. 84: 4762-4766). The β-lactamase gene isalso present to allow maintenance of plasmids in the host strain. Toallow genetic selection, the CAT structural gene from pJLS1021(Schottel, J. L., et al. (1984) Gene 28: 177-193) was amplified andplaced downstream of a constitutive trp^(c) promoter using PCR.Expression of the CAT gene in E. coli renders the cell resistant tochloramphenicol and the minimal inhibitory concentration, hereinafterreferred to as MIC, of chloramphenicol increases proportionally with theamount of CAT protein produced (Lee, K., et al. (1996) RNA 2: 1270-1285;Lee, K., et al. (1997) J. Mol. Biol. 269: 732-743) An overview of thesteps used to construct the system is shown in FIG. 2.

Selection of a new MBS-RBS pair. To isolate message bindingsite-ribosome binding site, hereinafter referred to as MBS-RBS,combinations that are nonlethal and efficiently translated only byplasmid-derived ribosomes, a random mutagenesis and selection schemewere used. In particular, the plasmid-encoded 16S MBS and CAT RBS wererandomly mutated using PCR so that the wild-type nucleotide at eachposition was excluded. An autoradiogram of sequencing gels withpRNA8-rMBS-rRBS is provided in FIG. 3. The resulting 2.5×10⁶ doublymutated transformants were induced for 3.5 hours in SOC mediumcontaining 1 mM IPTG and plated on Luria broth medium containing 100μg/mL ampicillin, 350 μg/mL chloramphenicol and 1 mM IPTG. To confirmthe presence of all three alternative nucleotides at each mutatedposition, plasmid DNA from approximately 2.0×10⁵ transformants wassequenced (FIG. 3).

Results

The data show that all of the nonexcluded nucleotides were equallyrepresented in the random pool. Of the 2.5×10⁶ transformants plated, 536survived the chloramphenicol selection. The efficiency of the selectedMBS-RBS combinations was determined by measuring the minimal inhibitoryconcentration, hereinafter referred to as MIC, of chloramphenicol foreach survivor in the presence and absence of inducer (FIG. 11) (Lee, K.,et al. (1996) RNA 2: 1270-1285; Lee, K., et al. (1997) J. Mol. Biol.269: 732-743). Nine of the isolates (1.7%) showed MIC in the presence ofinducer, which were lower than the 350 μg/mL concentration at which theywere selected. These were slow growing mutants that appeared after 48hours during the initial isolation. The MIC, however, were scored afteronly 24 hours. The MIC for 451 of the isolates (84.1%) were between 400and 600 μg/mL, and the remaining 76 clones (14.2%) were 600 μg/mL. Thedifference in chloramphenicol resistance between induced and uninducedcells (ΔMIC) is the amount of CAT translation by plasmid-derivedribosomes only. A specific interaction between plasmid-derived ribosomesand CAT mRNA was indicated in 79 (14.7%) of the clones, which showedfour- to eightfold increases in CAT resistance upon addition of IPTG(FIG. 11).

Based on these analyses, 11 clones were retained for additional study.The MBS and RBS in plasmids from these clones were sequenced and CATassays and growth curves were performed (FIGS. 4 and 12). Although awide range of inducibility was observed, there was no correlationbetween specificity and predicted free energy (ΔG^(°37)). Purines werepreferred in all of the MBS positions, but the RBS did not show thissort of selectivity. This can be explained partially by the observationthat the selected RBS can base pair with sequences adjacent to themutated region of 16S rRNA (Lee, K., et al. (1996)RNA 2: 1270-1285).

Growth curves were performed for all of the selected mutants andcompared with strains containing control constructs (FIG. 4). Only onemutant (IX24) is shown in FIG. 4, but all strains containing theselected MBS/RBS sequences showed the same pattern of growth as thismutant. Because of its induction profile, strain IX24 (containingplasmid pRNA100) was chosen for additional experimentation. To eliminatethe possibility that mutations outside the MBS and RBS had beeninadvertently selected, the DraIII and XbaI fragment containing the MBSand the KpnI and XhoI fragment containing the RBS sequence from pRNA100(FIG. 5) were transferred to pRNA9.

Specificity of the system. The rate of ribosome induction and the ratioof plasmid to chromosome-derived rRNA at each stage of growth weredetermined. For this, a pRNA100 derivative, pRNA104, which contains aC1192U mutation in 16S rRNA was constructed (Sigmund, C. D., et al.(1984) Nucleic Acids Res. 12: 4653-4663; Triman, K., et al. (1989) J.Mol. Biol. 209: 645-653) so that plasmid-derived rRNA could bedifferentiated from wild-type rRNA by primer extension. The C1192Umutation does not affect ribosome function in other expression systems(Sigmund, C. D., et al. (1984) Nucleic Acids Res. 12: 4653-4663;Makosky, P. C. et al. (1987) Biochimie 69: 885-889). To show that thesame is true in the present system, CAT activity was measured after 3hours induction with 1 mM IPTG in DH5 cells expressing pRNA100 orpRNA104 and the two were compared. In these experiments, no significantdifference between cells expressing pRNA104 (99.2±2.8%) or pRNA100(100%) was observed.

To determine the percentage of plasmid-derived ribosomes in cellscontaining the plasmid, total RNA was isolated from DH5 cells carryingpRNA104 before and after induction with IPTG and subjected to primerextension analysis (Lee, K., et al. (1997) J. Mol. Biol. 269: 732-743;Sigmund, C. D., et al. (1984) Nucleic Acids Res. 12: 4653-4663; Makosky,P. C. et al. (1987) Biochimie 69: 885-889). Maximum induction ofplasmid-derived ribosomes occurred 3 hours after induction at whichpoint they constituted approximately 40% of the total ribosome pool(FIG. 6). CAT activities in these cells paralleled induction ofplasmid-derived ribosomes and began to decrease 4 hours after induction,presumably due to protein degradation during stationary phase. Inuninduced cells, approximately 3% of the total ribosome pool containsplasmid-derived ribosomes because of basal level transcription from thelacUV5 promoter.

Optimization of the system. Chloramphenicol resistance in uninducedcells containing pRNA100 is 75 μg/mL (FIG. 13, MIC=100 μg/mL). Bymeasuring CAT resistance in a derivative of pRNA100 containing awild-type 16S rRNA gene, it was determined that approximately one-halfof this background activity was due to CAT translation by wild-typeribosomes (FIG. 13, pRNA100 1 wt MBS). The remaining activity inuninduced cells is presumably due to leakiness of the lacUV5 promoter(FIG. 6). The nucleotide sequence located between the RBS and the startcodon in mRNA affects translational efficiency (Calos, M. P. (1978)Nature 274: 762-765; Stormo, G. D., et al. (1982) Nucleic Acids Res. 10:2971-2996; Chen, H., et al. (1994) Nucl. Acids Res. 22: 4953-4957). InpRNA100, three of the nucleotides found in this region of the CAT mRNAare complementary with the 3′ terminus of wild-type E. coli 16S RNA(FIG. 11, pRNA100 1 wt MBS). To eliminate the possibility that this wascontributing to CAT translation in the absence of plasmid-encodedribosomes, four nucleotides in the CAT gene (underlined in FIG. 11) wererandomly mutagenized and screened to identify mutants with reducedtranslation by host ribosomes. A total of 2000 clones were screened inthe absence of plasmid-encoded ribosomes using pCAM9 and six poorlytranslated CAT sequences were isolated (FIG. 13). Next, the BamHIfragment of pRNA100 containing lacl^(q) and the rrnB operon was added,and MIC, CAT assays and growth curves were performed on cells expressingthese constructs (data not shown).

Based on these data, pRNA122 was chosen because it produced a slightlybetter induction profile than the others (FIGS. 11 and 23). Translationof the pRNA122 CAT message by wild-type ribosomes (FIG. 11, pRNA122 1 wtMBS) produces cells that are sensitive to chloramphenicol concentrations<10 μg/mL. In the presence of specialized ribosomes (FIG. 13, pRNA122),the background chloramphenicol MIC is between 40 and 50 μg/mL and theMIC for induced cells is between 550 and 600 μg/mL, producing anapproximately 13-fold increase in CAT expression upon induction inpRNA122. Induction of the rrnB operon in pRNA100 produces only aneightfold increase.

Use of the system. To test the system, the effects of nucleotidesubstitutions at the sole pseudouridine in E. coli 16S rRNA, located atposition 516 were examined. Because pseudouridine and U form equallystable base pairs with adenosine (Maden, B. E. (1990) Prog. Nucleic AcidRes. Mol. Biol. 39: 241-303), mutations at A535 were also constructed todetermine whether the potential for base pair formation between thesetwo loci affected ribosome function. The mutations were constructedinitially in a pUC19 (Yanisch-Perron, C., et al. (1985) Gene 33:103-119) derivative containing the 16S RNA gene, p16ST, as shown in FIG.7 and then transferred to pRNA122 for analysis. This two-step processwas used, because the SacII restriction site located between the twomutated positions is unique in pRNA16ST and is not unique in pRNA122.The effect of the mutations in pRNA122 on protein synthesis in vivo wasdetermined by measuring the MIC and CAT activity of the mutant cells(FIG. 8). At position 516, ribosomes containing the single transitionmutation, pseudouridine516C, produced approximately 60% of the amount offunctional CAT protein produced by wild-type ribosomes. The transversionmutations, pseudouridine516A or pseudouridine516G, however, reducedribosome function by >90%. All of the single mutations at position 535retained >50% of the function of wild-type ribosomes. To examine thepossibility that the potential for base pairing between positions 516and 535 is necessary for ribosome function, all possible mutationsbetween these loci were also constructed and analyzed (FIG. 8). Thesedata show that all of the double mutants were inactive (10% or less ofthe wild-type) regardless of the potential to base pair. To examine thereasons for loss of function in the 516 mutants, ribosomes from cellsexpressing single mutations at position 516 were fractionated by sucrosedensity gradient centrifugation and the 30S and 70S peaks were analyzedby primer extension to determine the percentage of plasmid-derived 30Ssubunits present. The data in FIG. 14 show a strong correlation betweenribosome function and the presence of plasmid-derived ribosomes in the70S ribosomal fraction, indicating that mutations at positions 516affect the ability of the mutant 30S subunits to form 70S ribosomes.

The references cited in Example 4 may be found in Lee, K., et al.Genetic Approaches to Studying Protein Synthesis: Effects of Mutationsat Pseudouridine516 and A535 in Escherichia coli 16S rRNA. Symposium:Translational Control: A Mechanistic Perspective at the ExperimentalBiology 2001 Meeting (2001) and at Lee, K. et al. (2001) GeneticApproaches to Studying Protein Synthesis: Effects of Mutations atPseudouridine516 and A535 in Escherichia coli 16S rRNA. J. Nutrition 131(11):2994-3004.

Example 5 In Vivo Determination of RNA Structure-Function Relationships

Materials and Methods

Reagents. Restriction enzymes, ligase, AMV reverse transcriptase andcalf intestine alkaline phosphatase were from New England Biolabs andfrom Gibco-BRL. Sequenase modified DNA polymerase, nucleotides andsequencing buffers were from USB/Amersham. Oligonucleotides weresynthesized on-site using a Beckman Oligo 1000 DNA synthesizer. AmplitaqDNA polymerase and PCR reagents were from Perkin-Elmer-Cetus.[³H]Chloramphenicol (30.1 Ci/mmol) was from Amersham and [α-³⁵S]dATP(1000 Ci/mmol) was from New England Nuclear. Other chemicals were fromSigma.

pRNA122. The key features of this construct are: (1) it contains a copyof the rrnB operon from pKK3535 (Brosius. J., et al. (1981) Plasmid6:112-118.) under transcriptional regulation of the lacUV5 promoter; (2)it contains a copy of the lactose repressor allele lacl^(q) (Calos, M.P. (1978) Nature 274:762-769; (3) the chloramphenicol acetyltransferasegene (cam) is present and transcribed constitutively from a mutanttryptophan promoter, trp^(c) (de Boer, H. A., et al. (1983) Proc. Natl.Acad. Sci. USA 80:21-25); (4) the RBS of the CAT message has beenchanged from the wild-type, 5′-GGAGG to 5′-AUCCC, and the MBS of the 16SrRNA gene has been changed to 5′-GGGAU; and (5) the β-lactamase gene ispresent to allow maintenance of plasmids in the host strain.

Bacterial strains and media. Plasmids were maintained and expressed inE. coli DH5 (supE44, hsdR17, recA1, endA1, gyrA96, thi-1; Hanahan, D.(1983) J. Mol. Biol. 166:557-580). Cultures were grown in LB medium(Luria, S. E. & Burrous, J. W. (1957) J. Bacteriol. 74:461-476) or LBmedium containing 100 μg/ml ampicillin (LB-Ap100). To induce synthesisof plasmid-derived rRNA from the lacUV5 promoter, IPTG was added to afinal concentration of 1 mM at the times indicated in each experiment.Strains were transformed by electroporation (Dower, W. J., et al. (1988)Nucl. Acids Res. 16: 6127) using a Gibco-BRL Cell Porator. Unlessotherwise indicated, transformants were grown in SOC medium (Hanahan,1983, supra) for one hour prior to plating on selective medium to allowexpression of plasmid-derived genes.

Chloramphenicol acetyltransferase assays. CAT activity was determinedessentially as described (Nielsen, D. A. et al. (1989) Anal. Biochem.60:191-227). Cultures for CAT assays were grown in LB-Ap100. Briefly,0.5 ml aliquots of mid-log cultures (unless otherwise indicated) wereadded to an equal volume of 500 mM Tris-HCl (pH8) and lysed using 0.01%(w/v) SDS and chloroform (Miller, J. H. (1992) A Short Course inBacterial Genetics, (Miller, J. H., ed.), pp. 71-80, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The resulting lysate waseither used directly or diluted in assay buffer prior to use. Assaymixtures contained cell extract (5 μl or 10 μl), 250 mM Tris (pH 8), 214μM butyryl-coenzyme A (Bu-CoA), and 40 μM [³H]chloramphenicol in a 125μl volume. Two concentrations of lysate were assayed for one hour at 37°C. to ensure that the signal was proportional to protein concentrations.The product, butyryl-[³H]chloramphenicol was extracted into2,6,10,14-tetramethylpentadecane:xylenes (2:1) and measured directly ina Beckman LS-3801 liquid scintillation counter. Blanks were preparedexactly as described above, except that uninoculated LB medium was usedinstead of culture.

Minimum inhibitory concentration determination. MICs were determined bystandard methods in microtiter plates or on solid medium. Overnightcultures grown in LB-Ap100 were diluted and induced in the same mediumcontaining 1 mM IPTG for three hours. Approximately 10⁴ induced cellswere then added to wells (or spotted onto solid medium) containingLB-Ap100+ IPTG (1 mM) and chloramphenicol at increasing concentrations.Cultures were grown for 24 hours and the lowest concentration ofchloramphenicol that completely inhibited growth was designated as theMIC.

Random mutagenesis and selection. Random mutagenesis of the 790 loop wasperformed essentially by the method of Higuchi (1989) using PCR andcloned in pRNA122 using the unique BglII and DraIII restriction sites(Higuchi, R. (1989) PCR Technology (Erlich, H. A., ed.), pp. 61-70,Stockton Press, New York) (FIG. 18). For each set of mutations, fourprimers were used: two “outside” primers and two “inside” primers. Thetwo outside primers were designed to anneal to either side of the BglIIand DraIII restriction sites in pRNA122 (FIG. 2). These primers were16S-DraIII, 5′-GACAATCTGTGTGAGCACTA-3′ (SEQ ID NO:239) and 16S-535,5′-TGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGT-3′ (SEQ ID NO:240). The insideprimers were 16S-786R, 5′-CCTGTTTGCTCCCCACGCTTTCGCACCTGAGCG-3′ (SEQ IDNO:241) and 16S-ASS-3,5′-CTCAGGTGCGAAAGCGTGGGGAGCAAACAGG CCTGGTAGTCCACGCC GTAA-3′ (SEQ ID NO:242) (N=A, T, C and G). Thus, 4⁹=262,144possible combinations were created, with the exception of 320 sequencesthat were eliminated because they formed either BglII or DraIIIrecognition sites (256 BglII sites and 64 DraIII sites).

Transformants were incubated in SOC medium containing 1 mM IPTG for fourhours to induce rRNA synthesis and then plated on LB agar containing 100μg/ml chloramphenicol. A total of 2×10⁶ transformants were platedyielding approximately 2000 chloramphenicol-resistant survivors. Next,736 of these survivors were randomly chosen and assayed to determine theMIC of chloramphenicol necessary to completely inhibit growth in cellsexpressing mutant ribosomes. From this pool, 182 transformants with MICsgreater than 100 μg/ml were randomly selected and sequenced.

Site-directed mutation of positions 787 and 795. Mutations at positions787 and 795 were constructed as described above for the random mutants,except that the inside primers were 16S-786R (see above) and16S-ASS-4,5′-CTCAGGTGCGAAAGCGTGGGGAGCAAACAGGNTTAGATANCCTGGTAGTCCACGCCGTAA-3′ (SEQ ID NO:243) (N=A, T, C and G). Transformants wereselected on LB-Ap100 agar plates and grouped according to their MICs forchloramphenicol. Representatives from each group were then sequenced toidentify the mutations.

Primer extension. To determine the ratio of plasmid tochromosome-derived rRNA, 30S and 70 S ribosomes were isolated from 200ml of induced, plasmid containing cells by the method of Powers & Noller(1991). The purified RNA was then used in primer extension experiments(Triman, K., et al. (1989) J. Mol. Biol. 209:643-653). End-labeledprimers complementary to sequences 3′ to the 788 and 795 mutation siteswere annealed to rRNA from induced cells and extended through themutation site using AMV reverse transcriptase. The primers used were:16S-806R, 5′-GGACTACCAGGGTATCT-3′ (SEQ ID NO:244); 16S-814R,5′-TACGGCGTGGACTACCA-3′ (SEQ ID NO:245). For wild-type pRNA122ribosomes, position 1192 in the 16S RNA gene was changed from C to U andprimers were constructed as described above (Triman et al., 1989,supra). This mutation has previously been shown not to affect subunitassociation (Sigmund, C. D., et al. (1988) Methods Enzymol.164:673-689). The extension mixture contained a mixture of threedeoxyribonucleotides and one dideoxyribonucleotide. The cDNAs wereresolved by PAGE and the ratios of mutant to non-mutant ribosomes weredetermined by comparing the amount of radioactivity in each of the twobands.

Oligoribonucleotide synthesis. Oligoribonucleotides were synthesized onsolid support with the phosphoramidite method (Capaldi, D. & Reese, C.(1994) Nucl. Acids Res. 22:2209-2216) on a Cruachem PS 250 DNA/RNAsynthesizer. Oligomers were removed from solid support and deprotectedby treatment with ammonia and acid following the manufacturer'srecommendations. The RNA was purified on a silica gel Si500F TLC plate(Baker) eluted for five hours with n-propanol/ammonia/water (55:35:10,by vol.). Bands were visualized with an ultraviolet lamp and the leastmobile band was cut out and eluted three times with 1 ml of purifiedwater. Oligomers were further purified with a Sep-pak C-18 cartridge(Waters) and desalted by continuous-flow dialysis (BRL). Purities werechecked by analytical C-8 HPLC (Perceptive Biosystems) and were greaterthan 95%.

Experimental Procedures

Sequence analysis of functional mutants. Random mutations wereintroduced simultaneously at all nine positions (787 to 795) in the 790loop. Functional (chloramphenicol-resistant) mutants were then selectedin E. coli DH5 cells (Hanahan, 1983, supra) and the effects of thesemutations on ribosome function were determined A total of 182 mutantsthat retained chloramphenicol resistance were randomly selected andsequenced. Wild-type 790-loop sequences were obtained from 81 of thesequenced transformants, while the remaining 101 contained mutantsequences. One of the transformants was chloramphenicol-resistant in theabsence of inducer, presumably due to a spontaneous mutation in the CATgene, and was excluded from further analysis. Of 100 sequencedfunctional mutants, 14 were duplicates and four sequences occurred threetimes. Thus, 78 different, functional, 790-loop mutants were analyzed(FIG. 19). According to resampling theory, this distribution indicatesthat of the 4⁹=262,144 possible sequences, only 190 (standard deviation30) unique sequences exist in the pool of selected functional mutants.Of the 78 mutants, 44 contained four to six substitutions out of thenine bases mutated and 21 of these retained greater than 50% of thewild-type activity. The minimal inhibitory concentration (MIC) ofchloramphenicol for cells expressing wild-type rRNA from pRNA122 is 600μg/ml. MICs of the mutants ranged from 150 to 550 μg/ml with a mean of320 μg/ml (standard deviation 89). The median and mode were both 350μg/ml.

Functional 790-loop mutants showed strong nucleotide preferences at allmutated positions, except positions 788 and 792, which showed a randomdistribution (FIG. 20) but significant covariation. No mutations wereobserved at U789 or G791. Mutations at these positions, however, werepresent in mutants that were selected for loss of function (not shown).Thus, these nucleotides appear to be directly involved in ribosomefunction. U789 is strictly conserved among bacteria but is frequentlyC789 among other organisms (FIG. 20). Chemical protection studies haveshown that G791 is specifically protected from kethoxal modification in70 S ribosomes and polysomes (Brow, D. A. & Noller, H. F. (1983) J. Mol.Biol. 163: 112-118; Moazed, D. & Noller, H. F. (1986) J. Mol. Biol. 191:483-493); and by poly(U) (Moazed & Noller, 1986, supra) and that G791becomes more accessible to kethoxal modification when 30S subunits areconverted from the “inactive” to “active” conformation (Moazed et al.,1986, supra).

Purines were strongly selected at position 787 (97.4%) while A and, to alesser extent, C were preferred at position 790 (98.7%) and U wascompletely excluded at both positions. At both position 793 and 795, A,C and U were equally distributed but G was selected against. Adenine anduracil were preferred at position 794 (81.8%).

Non-random distribution of nucleotides among the selected functionalclones indicates that nucleotide identity affects the level of ribosomefunction. To examine this, the mean activities (MICs) of ribosomescontaining all mutations at a given position were compared bysingle-factor analysis of variance between ribosome function (MIC) andnucleotide identity at each mutated position. Positions that showed asignificant effect of nucleotide identity upon the level of ribosomefunction were 787 (P <0.001), 788 (P <0.05) and 795 (P<0.001). Theabsence of mutations at positions U789 and G791 in the functional clonesprevents statistical analysis of these positions but mutations at thesepositions presumably strongly affect ribosome function as well.

FIG. 20 shows a comparison of the selected functional mutants withcurrent phylogenetic data (R. Gutell, unpublished results; Gutell, R. R.(1994) Nucl. Acids Res. 22(17): 3502-3507; Maidak, B. L. et al. (1996)Nucl. Acids Res. 24: 82-85). While nucleotide preferences in theselected mutants are similar to those observed in the phylogenetic data,the mutant sequences selected in this study show much more variabilitythan those found in nature. This may be because all of the positions inthe loop were mutated simultaneously, allowing normally deleteriousmutations in one position to be compensated for by mutations at otherpositions, a process that is unlikely to occur in nature. In addition,none of the mutants was as functional as the wild-type, suggesting thatwild-type 790-loop sequences have been selected for optimal activity orthat other portions of the translational machinery have been optimizedto function with the wild-type sequence.

To identify potential nucleotide covariation within the loop, the paireddistribution of selected nucleotides was examined for goodness of fit.The most significant covariations were observed between positions 787and 795 (P <0.001) and between positions 790 and 793 (P <0.001). Forpositions 790 and 793, only eight double mutants were available foranalysis; therefore, the covariation observed between these positionsshould be regarded with caution. Position 788, which showed nonucleotide specificity, did show significant covariation with positions787 (P<0.01), 794 (P <0.01) and 795 (P <0.01).

Analysis of site-directed mutations constructed at the base of the loop:

Functional analysis of mutations at positions 787 and 795. The observedcovariations among positions 787, 788 and 795 are particularlyinteresting, since nucleotide identity at these positions correlatedwith the level of ribosome function. Further analysis of nucleotides atpositions 787 and 795 revealed that 72 of the 78 functional mutants havethe potential to form mismatched base-pairs (A.C, G.U, A.A and G.G).Other mismatches, such as G.A and U.G, however, were not found. Inaddition, only four sequences with an A.U Watson-Crick pair and nosequences with a U.A, G.C or C.G pair were present, suggesting thatstrong base-pairs between these positions inhibit ribosome function.Therefore all possible nucleotide combinations at positions 787 and 795were constructed and analyzed without changing other nucleotides in the790 loop. Ribosome function of the mutants (FIG. 21) varied from 84%(A.A) to 1% (C.G) of the wild-type. As predicted by analysis of the poolof functional random mutants, site-directed mutants with G.C, C.G andU.A Watson-Crick pairs between positions 787 and 795 were stronglyinhibitory.

Results

These data suggest that strong pairing between nucleotides at positions787 and 795 inhibits ribosome function. In addition, some of thesite-directed substitutions at positions 787 and 795 that producedfunctional ribosomes were largely excluded from the pool of mutants inwhich all of the loop positions were mutated simultaneously (e.g. CC,CU, UU and UC). The observed nucleotide preferences at positions 787 and795 in the selected random pool presumably reflect interaction ofnucleotides at these positions with other nucleotides in the loop. Thisis consistent with our findings of extensive covariations among thesesites.

Perturbations of the 790 loop have been shown to affect ribosomalsubunit association (Herr, W., et al. (1979) J. Mol. Biol. 130: 433-449;Tapprich, W. & Hill, W., (1986) Proc. Natl. Acad. Sci. USA 83: 556-560;Tapprich, W., et al. (1989) Proc. Natl. Acad. Sci. USA 86: 4927-4931).Therefore several of the 787 to 795 mutants were tested for theirability to form 70 S ribosomes. Ribosomes were isolated from selectedmutants and the distribution of mutant ribosomes in both the 70 S and30S peaks was determined by primer extension (FIG. 21). These data showthat CAT activity correlates with the presence of mutant 30S subunits inthe 70 S ribosome pool. Thus, loss of function may be due to theinability of mutant 30S and 50 S subunits to associate. Anotherexplanation for this observation is that the mutations may directlyaffect a stage of the protein synthesis process prior to subunitassociation, such as initiation, which prevents subsequent steps fromoccurring. Other mutations in the 16S rRNA have been identified forwhich this appears to be the case (Cunningham, P., et al. (1993)Biochemistry 32: 7172-7180).

The references cited in Example 5 may be found in Lee, K. et al., J.Mol. Biol. 269: 732-743 (1997), expressly incorporated by referenceherein.

Example 6 Construction of a Hybrid Construct

A plasmid construct of the present invention identified as the hybridconstruct, is set forth in FIGS. 17 and 25. This hybrid constructcontains a 16S rRNA from Mycobacterium tuberculosis. The specific siteson the hybrid construct are as follows: the part of rRNA from E. colirrnB operon corresponds to nucleic acids 1-931; the part of 16S rRNAfrom Mycobacterium tuberculosis rrn operon corresponds to nucleic acids932-1542; the 16S MBS GGGAU corresponds to nucleic acids 1536-1540; theterminator T1 of E. coli rrnB operon corresponds to nucleic acids1791-1834; the terminator T2 of E. coli rrnB operon corresponds tonucleic acids 1965-1994; the replication origin corresponds to nucleicacids 3054-2438; the bla (β-lactamase; ampicillin resistance)corresponds to nucleic acids 3214-4074; the GFP corresponds to nucleicacids 5726-4992; the GFP RBS (ribosome binding sequence) AUCCCcorresponds to nucleic acids 5738-5734; the trp^(c) promoter correspondsto nucleic acids 5795-5755; the trp^(c) promoter corresponds to nucleicacids 6270-6310; the CAT RBS (ribosome binding sequence) AUCCCcorresponds to nucleic acids 6327-6331; the cam (chloramphenicolacetyltransferase; CAT) corresponds to nucleic acids 6339-6998; thelacl^(q) promoter corresponds to nucleic acids 7307-7384; the lacl^(q)(lac repressor) corresponds to nucleic acids 7385-8467; and the lac UV5promoter corresponds to nucleic acids 8510-8551.

All references cited herein are expressly incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for identifying functional mutant ribosomes comprising: (a)transforming a set of host cells with a set of plasmids, each plasmidcomprising a mutant rRNA gene and a selectable marker gene; wherein saidmutant rRNA gene comprises at least one mutation in addition to a mutantAnti-Shine-Dalgarno sequence; and said selectable marker gene comprisesa mutant Shine-Dalgarno sequence; and wherein said mutantAnti-Shine-Dalgarno and said mutant Shine-Dalgarno sequence are amutually compatible pair; thereby forming a set of transformed hostcells; (b) isolating from the set of transformed host cells those hostcells which express the selectable marker gene product; and (c)sequencing the mutant rRNA gene from each host cell isolated in step(b), thereby identifying functional mutant ribosomes, wherein thefunctional mutant ribosomes comprise functionally important targetregions of interest, which comprise nucleotide sequences of one or morenucleic acids or nucleotide motifs which are conserved in each mutantrRNA gene sequenced.
 2. The method of claim 1 wherein said mutant rRNAgene is a mutant E. coli 16S rRNA gene and said selectable marker geneis a green fluorescent protein gene.
 3. A method for identifyingfunctional mutant ribosomes that may be suitable as drug targetscomprising: identifying functional mutant ribosomes according to themethod of claim 1, thereby identifying functionally important targetregions of interest, wherein said regions of interest comprise sequencesof one or more nucleic acids or nucleotide motifs which are conserved ineach mutant rRNA gene sequenced; (d) generating a second plurality ofmutant rRNA genes wherein said target regions of interest are mutated;and each of said rRNA genes of said second plurality of mutant rRNAgenes further comprises a second mutant Anti-Shrine-Dalgarno sequence;(e) inserting the second plurality of mutant rRNA genes comprising themutated regions of interest from step (d) into a second plurality ofplasmids; wherein said second plurality of plasmids further comprise asecond genetically engineered gene which encodes a second selectablemarker having a second mutant Shine-Dalgarno sequence, wherein thesecond mutant Anti-Shine-Dalgarno and the second mutant Shine-Dalgarnosequence are a mutually compatible pair; (f) transforming a second setof host cells with the plasmids from step (e), thereby forming a secondset of transformed host cells; (g) isolating from the second set oftransformed host cells of step (f) those host cells which express thesecond selectable marker gene product; and (h) sequencing the rRNA genefrom each host cell isolated in step (g), thereby identifying functionalmutant ribosomes that may be suitable as drug targets.
 4. The method ofclaim 1, wherein said mutant rRNA gene is selected from the rRNA genesof Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa,Salmonella typhi, Yersenia pestis, Staphylococcus aureus, Streptococcuspyogenes, Enterococcus faecalis, Chlamydia trachomatis, Saccharomycescerevesiae, Candida albicans, and trypanosomes.
 5. The method of claim1, wherein said mutant rRNA gene is a 16S rRNA gene.
 6. The method ofclaim 1, wherein the selectable marker is selected from the groupconsisting of chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), and mixtures thereof.
 7. The method of claim 1, whereinthe selectable marker gene is green fluorescent protein.
 8. The methodof claim 1, wherein said mutant Shine-Dalgarno sequence is selected fromthe group consisting of the sequences set forth in SEQ. ID. NOS: 24-159;and said mutant Anti-Shine-Dalgarno sequence is selected from the groupconsisting of the sequences set forth in SEQ. ID. NOS: 24-159.
 9. Themethod of claim 2, wherein said mutant Shine-Dalgarno sequence isselected from the group consisting of the sequences set forth in SEQ.ID. NOS: 24-159; and said mutant Anti-Shine-Dalgarno sequence isselected from the group consisting of the sequences set forth in SEQ.ID. NOS: 24-159.
 10. The method of claim 3, wherein said first mutantrRNA gene is selected from the rRNA genes of Escherichia coli,Mycobacterium tuberculosis, Pseudomonas aeruginosa, Salmonella typhi,Yersenia pestis, Staphylococcus aureus, Streptococcus pyogenes,Enterococcus faecalis, Chlamydia trachomatis, Saccharomyces cerevesiae,Candida albicans, and trypanosomes.
 11. The method of claim 3, whereinsaid first mutant rRNA gene is a 16S rRNA gene.
 12. The method of claim3, wherein the first selectable marker is selected from the groupconsisting of chloramphenicol acetyltransferase (CAT), green fluorescentprotein (GFP), and mixtures thereof.
 13. The method of claim 3, whereinthe first selectable marker gene is green fluorescent protein.
 14. Themethod of claim 3, wherein the second selectable marker is selected fromthe group consisting of chloramphenicol acetyltransferase (CAT), greenfluorescent protein (GFP), and mixtures thereof.
 15. The method of claim3, wherein the second selectable marker gene is green fluorescentprotein.
 16. The method of claim 3, wherein said first mutantShine-Dalgarno sequence is selected from the group consisting of thesequences set forth in SEQ. ID. NOS: 24-159; and said first mutantAnti-Shine-Dalgarno sequence is selected from the group consisting ofthe sequences set forth in SEQ. ID. NOS: 24-159.
 17. The method of claim3, wherein said second mutant Shine-Dalgarno sequence is selected fromthe group consisting of the sequences set forth in SEQ. ID. NOS: 24-159;and said second mutant Anti-Shine-Dalgarno sequence is selected from thegroup consisting of the sequences set forth in SEQ. ID. NOS: 24-159.